Abstract
A frame of reference for the genetic profiles of grapevine cultivars whose cultivation is permitted in Castilla La Mancha (Spain) was established by analyzing 26 nuclear and five chloroplast microsatellite loci. The aim was to validate plant material in such a way as to identify those errors of nomenclature (synonyms and homonyms) that are common in grapevine and that can cause administrative problems and then to search for possible parent-offspring combinations. Results showed no differentiated genetic profile for cv. Verdoncho, which is a synonym of Pardina/Jáen Blanco. The Alarije cv. has a dual legal status, as it coincides genetically with Torrontés. There are also several cases of homonyms affecting the denominations Albillo, Coloraíllo, and Moravia Dulce. Each of these embraces a group of different cultivars, some of which have not been described previously, such as Albillo Dorado. Results confirm the origin of cultivars Albillo Dorado, Coloraíllo, and Moribel from a cross between Tempranillo and Moravia Dulce, with Moravia Dulce the female parent for all.
Castilla La Mancha, a region with a strong winemaking tradition, covers an area of 79,500 km2 in the center of the Iberian Peninsula and has approximately 490,824 ha of vineyard, comprising 49% of the total for Spain, 14% of the total for Europe, and 7% of the total for the world. The grapevine heritage of the region is comprised of dozens of cultivars, dominated by Airén (220,420 ha) and Macabeo (9,316) for white and Tempranillo or Cencibel (73,931), Bobal (41,045), Garnacha Tinta (20,390), and Monastrell (19,905) for red (Consejería de agricultura y medio ambiente 2011). The remaining vineyard area is planted with other cultivars, some of which are found only locally. The emergence of such local cultivars, whose particular properties are not yet known, is a consequence of the large size of the region, which presents highly diverse edaphic-climatic conditions, and the high intensity of cultivation.
The current regulation of potential grapevine production specifies the winemaking cultivars that may be grown in each region and classifies them as recommended or authorized. For Castilla La Mancha, 15 recommended cultivars are listed, seven red and eight white, and 31 authorized cultivars, 17 red and 14 white. The latest amendment was introduced by Royal Decree 461/2011 of 1 April regulating potential grapevine production. This research group has extensive experience in grapevine prospecting, characterization, and identification using molecular tools, thus making it possible to validate plant material and to help detect those errors of nomenclature (synonyms and homonyms) that are common in grapevine and that give rise to administrative problems.
This article seeks to establish a reference framework for the genetic profiles of the grapevine cultivars whose cultivation is permitted in the Castilla La Mancha region. To that end, 26 nuclear and five chloroplast microsatellite markers were analyzed, including six (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79) that correspond to OIV 801 to 806 descriptors (OIV 2009).
Materials and Methods
A total of 144 grapevine accessions belonging to the 46 cultivars authorized or recommended for winemaking in Castilla La Mancha were considered for this study (Supplemental Table 1). These cultivars were directly obtained from the germplasm collection maintained at the Instituto de la Vid y el Vino de Castilla La Mancha (IVICAM).
For analysis of the microsatellite regions, DNA was extracted from vine shoots, as previously described (Vilanova et al. 2009), and 26 SSR loci were analyzed in order to obtain information from each of the 19 chromosomes of the vine. Six of the loci correspond to OIV 801 to 806 descriptors (OIV 2009): VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79. The other loci were VVMD21, VVMD24, VVMD25, VVMD28, VVMD32 (Bowers et al. 1999b); VrZAG21, VrZAG64, VrZAG67, VrZAG83 (Sefc et al. 1999); VVIB01, VVIH54, VVIN16, VVIN73, VVIP31, VVIP60, VVIQ52, VVIV37, VVIV67 (Merdinoglu et al. 2005); VMC1b11 (Welter et al. 2007); and VMC4F3.1 (Di Gaspero et al. 2000).
The forward primer from each pair was fluorescently labeled to allow detection, using 6-FAM (blue), VIC (green), PET (red), and NED (yellow) (Applied Biosystems, Foster City, CA). Three previously optimized multiplex PCR reactions were performed to obtain a similar quantity of amplifications for all the microsatellite markers. PCR reactions were carried out in a GeneAmp PCR System 9700 thermocycler (PE Applied Biosystems), in 10 μL of a mixture containing 5 to 10 ng DNA (Qiagen multiplex PCR Kit 1X; Qiagen, Hilden, Germany) and different amounts of each primer pair depending on the set (Supplemental Table 2). PCR conditions were 95°C for 15 min, 30 cycles of 30 sec at 95°C, 90 sec at 55°C, and 60 sec at 72°C, and a final extension of 30 min at 72°C.
Five chloroplast loci were also amplified—CCMP3, CCMP5, and CCMP10 (Weising and Gardner 1999) and ccSSR9 and ccSSR14 (Chung and Staub 2003)—under the conditions described by Ibáñez et al. (2009). The amplified products were separated by capillary electrophoresis and analyzed by fluorescence using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The sizes of the fragments, expressed in base pairs (bp), were calculated with GeneMapper software, using GeneScan 500 LIZ (Applied Biosystems) as internal size standard.
The genotypes of all the accessions in this study were compared with various databases (Sánchez-Escribano et al. 1999, Sefc et al. 2000, Martín et al. 2003, Ibáñez et al. 2003, This et al. 2004, Jiménez-Cantizano et al. 2006, Yuste et al. 2006, Ibáñez et al. 2009, Santana et al. 2010, BIOVID project [http://www.neiker.net/BT/], Grape 81 SSR Fingerprinting NCGR-Davis [www.ars.usda.gov/Main/docs.htm?docid=13743]).
The software Identity 1.0 (Wagner and Sefc 1999) was used to process data from the 26 SSRs and calculate the number and frequency of alleles, expected and observed heterozygosity values, frequency of null alleles, paternity exclusion probability, probability of identity, and the cumulative likelihood ratios for the proposed parentage (Vouillamoz et al. 2007).
Results
The 46 cultivars authorized and recommended for cultivation in Castilla La Mancha produced 46 different genotypes for the 26 nuclear microsatellite loci analyzed. The size of the alleles (Supplemental Table 3) and the chlorotype group to which they belong (Supplemental Table 4) were determined.
Differences found among the statistical parameters examined for the 26 SSR loci are reported (Table 1). The total number of alleles (NA) per locus ranged from 3 for locus VVIQ52 to 13 for locus VMC4F3.1, with an average of 8 alleles per locus. The expected heterozygocity (He) ranged from 48.7% for VVIN73 to 86.3% for VMC4F3.1, with an average of 75.3%, while the observed heterozygosity (Ho) ranged from 52.2% for VVIN73 and 91.3% for VMC4F3.1 and VVS2, with an average of 79.4%. Only four SSR loci presented a frequency of null alleles that was positive but small enough to be taken into account, and therefore accessions with only one allele per locus were treated as homozygotic rather than heterozygotic with a null allele.
The probability of identity (PI) ranged from 0.062 (VMC4F3.1) to 0.35 (VVIB01). The probability of finding identical genotypes among unrelated cultivars using these 26 SSR loci in combination was less than 10−22. These values are low enough to support the synonyms detected within the sample (Sefc et al. 2001). Likelihood ratios of proposed parentage versus any other two parents (46 cultivars at 26 microsatellites) were between 1014 and 1017 (Table 2), in the range of other likelihood ratios published in grapevine (Bowers et al. 1999a, Ibáñez et al. 2009).
Discussion
All accessions collected named as Verdoncho, an authorized white cultivar, corresponded to other cultivars. In particular, an accession from the germplasm collection of El Encín (P22-I30) was misnamed and corresponds to Pardillo. The remaining samples were identified as Pardina/Jaén Blanco. Given that the most widespread Verdoncho accession in the Castilla La Mancha region is identical to Pardina/Jaén Blanco, it is proposed to name the Verdoncho cultivar as a synonym of Pardina/Jaén Blanco and not as a new one.
The Alarije cv. was authorized recently. Moreover, this cultivar had already been classified as recommended in the region but under the name Torrontés (Fernández-González et al. 2007a), which is synonym. Alarije should therefore be the prime name included as a recommended cultivar with Torrontés as a synonym.
The Albillo cv. is a complex case of homonyms. As many as two cultivars with different genetic profiles are grown in Castilla La Mancha under the name Albillo. In the Méntrida Appellation of Origin (AO), Albillo Real is the typical cultivar, while in Manchuela AO, Albillo Dorado is typical; this cultivar is not contained in the list of commercial cultivars of grapes. Albillo Dorado represents a new genotype that has never been described according to the literature consulted. Albillo Dorado and Albillo Real should undoubtedly be differentiated and listed as distinct entities, since both are traditionally grown in the region.
A similar case is that of the recommended cv. Coloraíllo, a homonym denoting at least three different cultivars, two of which are new genotypes (G10 and G13) (Fernandez-González et al. 2007a). The case is further complicated by the fact that the name Coloraíllo is used erroneously in a general way to denote other cultivars such as Rojal Tinta and Teta de Vaca. Another homonym is Moravia Dulce, a name used to denote two different cultivars, one of which is a synonym of Crujidera and the other a newly described genotype (G9) (Fernández-González et al. 2007a, 2007b) named Moribel.
It is important to emphasize that the cultivars Garnacha Tinta (recommended) and Garnacha Peluda (authorized) as well as Tinto Velasco (recommended) and Tinto de la Pámpana Blanca (authorized) cannot be distinguished by analyzing microsatellite regions that are located in noncoding regions of the genome (Zulini et al. 2005). They differ only in some ampelographic characteristics such as leaf hair density (Muñoz-Organero et al. 2002). These cases of synonyms could be clones of the same cultivar, which show phenotypic differences (Walker et al. 2006).
Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplasts in Vitis vinifera and to characterize the chloroplast haplotypes present in winegrape cultivars of those species grown in Castilla La Mancha (Arroyo-García et al. 2002). Most of the cultivars (65%) carried chlorotype A (Supplemental Table 3), typical for Iberian and western European wine grapevines (Arroyo-García et al. 2006), followed by chlorotype D (26%), which is more frequently found in Central Europe. Chlorotype C was found at low frequency (7%), which is logical since this chlorotype was previously reported to be present at higher frequencies among table-grape cultivars from the Near and Middle East, and this work was limited to grapes for wine production. Finally, only Muscat of Alexandria belonged to chlorotype B, which was also the least common chlorotype for winegrapes according to Arroyo-García et al. (2006).
Microsatellite markers have proven suitable for consolidating grape cultivars and investigating the pedigree of cultivars (Bowers and Meredith 1997). Tempranillo and Moravia Dulce were confirmed as parent cultivars of Albillo Dorado, Coloraíllo, and Moribel (G9). Moreover, Moravia Dulce and Pardillo were described as the parents of Coloraíllo (G10). These four cultivars are from the Manchuela AO and the parents are also present in that area, so it is quite possible that the crosses occurred there. As chloroplast DNA is maternally inherited in Vitis, it can be said that Moravia Dulce was the female parent of Albillo Dorado, Coloraíllo, Moribel (G9), and Coloraíllo (G10), since the four displayed chlorotype D.
Conclusions
We have sought to establish a reference framework for the genetic profiles of the grapevine cultivars whose cultivation is permitted in the Castilla La Mancha region. We have also detected a number of new synonyms, such as Verdoncho/Jaén Blanco, some errors of denomination, such as Verdoncho/Pardillo, and homonyms affecting such groups, such as Coloraíllo, Moravia Dulce, and Albillo.
Acknowledgments
Acknowledgments: This research is part of a project entitled Prospecting, Identification, and Preservation of Minority Grapevine (Vitis vinifera L.) cultivars in Castilla La Mancha (PAI06-0026), funded by the Government of Castilla La Mancha.
Footnotes
-
Supplemental data is freely available with the online version of this article.
- Received March 2012.
- Revision received June 2012.
- Accepted July 2012.
- Published online December 1969
- ©2012 by the American Society for Enology and Viticulture
Literature Cited
Sign in for ASEV members
ASEV Members, please sign in at ASEV to access the journal online.
Sign in for Institutional and Non-member Subscribers
Log in using your username and password
Pay Per Article - You may access this article (from the computer you are currently using) for 2 day for US$10.00
Regain Access - You can regain access to a recent Pay per Article purchase if your access period has not yet expired.